STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and ...

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STK11/LKB1 Deficiency Promotes NeutrophilRecruitment and Proinflammatory CytokineProduction to Suppress T-cell Activity in the LungTumor MicroenvironmentShohei Koyama1,2, Esra A. Akbay2,3,Yvonne Y. Li2,3, Amir R. Aref2,3, Ferdinandos Skoulidis4,GritS.Herter-Sprie2,3,KevinA.Buczkowski3,YanLiu2,3,MarkM.Awad2,3,WarrenL.Denning4,LixiaDiao5, JingWang5, EdwinR. Parra-Cuentas6, Ignacio I.Wistuba6,Margaret Soucheray7,Tran Thai3, Hajime Asahina2,3, Shunsuke Kitajima3, Abigail Altabef3, Jillian D. Cavanaugh3,Kevin Rhee3, Peng Gao3, Haikuo Zhang2,3, Peter E. Fecci8,Takeshi Shimamura9, Matthew D.Hellmann10, John V. Heymach4, F. Stephen Hodi2,3, Gordon J. Freeman1,2, David A. Barbie2,3,Glenn Dranoff11, Peter S. Hammerman2,3, and Kwok-Kin Wong2,3,12

Abstract

STK11/LKB1 is among the most commonly inactivated tumorsuppressors in non–small cell lung cancer (NSCLC), especially intumors harboring KRAS mutations. Many oncogenes promoteimmune escape, undermining the effectiveness of immunothera-pies, but it is unclear whether the inactivation of tumor suppressorgenes, such as STK11/LKB1, exerts similar effects. In this study, weinvestigated the consequences of STK11/LKB1 loss on the immunemicroenvironment in a mouse model of KRAS-driven NSCLC.Genetic ablation of STK11/LKB1 resulted in accumulation of neu-trophils with T-cell–suppressive effects, alongwith a correspondingincrease in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lympho-cytes was also reduced in LKB1-deficient mouse and human

tumors. Furthermore, STK11/LKB1–inactivating mutations wereassociatedwith reduced expression of PD-1 ligand PD-L1 inmouseandpatient tumors aswell as in tumor-derived cell lines.Consistentwith these results, PD-1–targeting antibodies were ineffectiveagainst Lkb1-deficient tumors. In contrast, treating Lkb1-deficientmice with an IL6-neutralizing antibody or a neutrophil-depletingantibody yielded therapeutic benefits associated with reducedneutrophil accumulation and proinflammatory cytokine expres-sion. Our findings illustrate how tumor suppressor mutations canmodulate the immunemilieuof the tumormicroenvironment, andthey offer specific implications for addressing STK11/LKB1–mutat-ed tumorswithPD-1–targeting antibody therapies.Cancer Res; 76(5);999–1008. �2016 AACR.

IntroductionThe discovery of a series of oncogene driver mutations and the

concept of oncogene addiction has changed the therapeuticapproach for subsets of patients with non–small cell lung cancers(NSCLC; ref. 1). While this targeted approach for tumors withspecific kinase alterations has been successful, KRAS mutation isthemost common genetic alteration drivingNSCLCs and remainsrefractory to targeted treatment strategies.KRAS-mutatedNSCLCs

are genomically more complex than those harboring mutatedEGFR or EML4-ALK and the concurrent loss of key tumor sup-pressors such as TP53 or STK11 is common inKRAS-mutated lungadenocarcinomas.

STK11/LKB1 is inactivated in approximately one-third of theKRAS-mutated lung adenocarcinomas, a frequency compara-ble with TP53 loss in this background, though STK11 and TP53mutations rarely overlap in KRAS-mutant lung tumors (2).

1Department of Medical Oncology and Cancer Vaccine Center, DanaFarber Cancer Institute, Boston, Massachusetts. 2Department ofMedicine, Brigham and Women's Hospital and Harvard MedicalSchool, Boston, Massachusetts. 3Department of Medical Oncology,Dana Farber Cancer Institute, Boston, Massachusetts. 4Departmentof Thoracic/Head and Neck Medical Oncology, The University ofTexas MD Anderson Cancer Center, Houston, Texas. 5Department ofBioinformatics and Computational Biology, The University of TexasMDAnderson Cancer Center, Houston, Texas. 6Department of Trans-lational Molecular Pathology, The University of Texas MD AndersonCancer Center, Houston, Texas. 7University of California, San Fran-cisco, San Francisco, California. 8Division of Neurosurgery, Depart-ment of Surgery, Duke University Medical Center, Durham, NorthCarolina. 9Department of Molecular Pharmacology and Therapeu-tics, Oncology Research Institute, Loyola University Chicago, Illi-nois. 10Department of Medicine, Memorial Sloan Kettering CancerCenter, New York, New York. 11Novartis Institutes for BioMedicalResearch, Cambridge, Massachusetts. 12Belfer Institute for Applied

Cancer Science, Dana Farber Cancer Institute, Boston,Massachusetts.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

S. Koyama, E.A. Akbay, and Y.Y. Li contributed equally to this article.

Corresponding Authors: Kwok-Kin Wong, Dana-Farber Cancer Institute, 44Binney St, Boston, MA 02115. Phone: 617-582-6084; Fax: 617-582-7839; E-mail:[email protected]; Peter Hammerman, Novartis Institutes for BioMedicalResearch, 250 Massachusetts Avenue, Cambridge, MA, 02139. E-mail:[email protected]; andGlennDranoff, Novartis Institutes forBioMedical Research, 250 Massachusetts Avenue, Cambridge, MA, 02139.E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-1439

�2016 American Association for Cancer Research.

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Lkb1-deficient Kras-mutated (Kras/Lkb1) tumors show a moreinvasive and metastatic phenotype with significantly reducedsurvival (3), and differential drug sensitivities as comparedwith Kras-mutant Lkb1-wild-type tumors and Kras-mutatedcompound Tp53-deficient animals (4). A more metastaticphenotype in KRAS/LKB1 tumors has also been described inclinical studies (5, 6).

Recent clinical trials in NSCLC have demonstrated response toimmune checkpoint blockade and nominated predictive markersfor the efficacy of specific immunotherapies (7–9). Our previouswork suggests that oncogenes impact immune evading mechan-isms by directly activating immune checkpoints (10). Immuneevasion can also be achieved by the release of proinflammatorycytokines into the tumor microenvironment that play an impor-tant role in promoting tumor growth, metastasis, and immunesuppression (11, 12). Previouswork has shown thatKras-mutatedtumors display activation of the noncanonical IkappaB kinaseTBK1 (13, 14), and the activation of this signaling pathwayinduces several proinflammatory cytokines, such as IL6 andCXC-chemokine ligands. Myeloid cells, especially tumor-associ-ated macrophages (TAM) and neutrophils (TAN), support tumorcell proliferation and impede host immune surveillance throughcytokine production and cell–cell interactions (15).

To elucidate how Stk11/Lkb1 (hereafter referred to as Lkb1 in themouse model) loss affects the inflammatory phenotype in Kras-driven lung cancer, we compared immune cell populations andcytokine/chemokine profiles among Kras and Kras/Lkb1 mouselung cancer models. We found that neutrophil-attracting solublefactors and neutrophil numbers were significantly increased andboth T-cell numbers and function were significantly decreased inLkb1-deficient tumors. Moreover, Lkb1 loss of function negativelyimpacted PD-L1 expression in lung tumor cells in mouse andhuman tumors and cell lines. By depleting the neutrophils inKras/Lkb1–mutant mice, T-cell numbers and function were significant-ly improved affirming the immunosuppressive properties of thiscell type. Finally, we functionally validated the therapeutic utilityof blocking the cytokine feedback loop with a neutralizing anti-IL6 antibody, which resulted in an increase of T-cell numbersand function. Together, the results suggest that in the Lkb1-deficient tumors, immune evasion is achieved through suppres-sive myeloid cells and aberrant cytokine production and not thePD-1:PD-L1 interaction.

Materials and MethodsMurine cell line and in vivo studies

Mouse strains were described previously (3). Mice were dosedwith 200 mg of IL6-neutralizing antibody (MP5-20F3, BioXcell),anti Ly-6G/Gr-1 antibody (RB6-8C5, BioXcell), PD-1–blockingantibody (clone 29F.1A12), and isotype controls (BioXcell) threetimes a week via intraperitoneal injections. MRI quantificationwas performed as described previously (10). Murine cell linesbearing mutated Kras and p53-loss (Kras/p53:KP) and mutatedKras and both p53 and Lkb1-loss (Kras/p53/Lkb1:KPL) were estab-lished and characterized as described previously (16). Recombi-nant mouse IL1a was from PeproTech.

Immune cell isolation, analysis, and sortingLung cell isolation, mononuclear cell enrichment, and charac-

terization of immune cell populations in murine tissue sampleswere described previously (10). Total cell count was divided bytumor-bearing lung weight utilized for each assay. Antibodies are

listed in Supplementary Methods. Intracellular staining for Ki-67,IFNg , CTLA-4, FOXP3, and LGALS9 was performed according tothe manufacturer's protocol (eBioscience and BD Biosciences).Sorting of tumor cells (CD45�EpCAMþ) and neutrophils(CD45þCD11bþLy-6Gþ) was performed on a BD FACSAria II.Gating methods for immune analysis and sorting are mentionedin Supplementary Methods.

Sample preparation for RNA sequencingRNA isolation from sorted cells was performed using the Pico-

Pure RNA Isolation Kit (Life Technologies) according to the man-ufacturer's protocol. Ten to 100 ng of total RNA was used as inputfor the generation libraries using the Nugen Ovation Kit. Librarieswerequality controlledonanAgilenthigh sensitivityDNAchipandsequencing of pooled libraries was performed on the IlluminaHiSeq platform to a minimum depth of 30 million reads.

Mouse RNA sequencing and patient tumor gene expression andproteomic data analysis (CCLE, TCGA, and PROSPECT)

For details, see Supplementary Methods.

Preparation of isogenic human cell lines and IL1-a stimulationHuman lung cancer cell lines were obtained from ATCC and

used prior to 6 months of passage in culture and not furtherauthenticated. Short hairpin RNA constructs and stable isogeniccell lines were established as described previously (17). Recom-binant human IL1a was from PeproTech (18).

IHCIHC for TUNEL and Ki-67 was performed as previously

described (19). For the PROSPECT samples, 4-mm thick tissuesections were stained using an automated staining system (LeicaBond Max, Leica Microsystems), according to the standard pro-tocols. The38 Aperio Image Analysis Toolbox (Aperio, LeicaMicrosystems) was used for digital analysis of images obtainedfrom scanned slides. PDL1 clone E1L3N from Cell SignalingTechnology, CD3 A0452 from Dako, and CD8 C8/144B fromThermo Scientific were used.

Western blottingTumor nodules resected from Kras and Kras/Lkb1 mice were

homogenized in RIPA buffer and proteinase inhibitor (CellSignaling Technology). Western blotting was performed asdescribed previously (18) with anti pSTAT3, STAT3, LKB1, andactin antibodies (Cell Signaling Technology).

Measurement of soluble factor concentrations inbronchoalveolar lavage fluids from mice and culturesupernatants from murine and human cell lines

For details, see Supplementary Methods.

Statistical analysisAll numerical data are presented as mean � SD. Data were

analyzed using two-tailed unpaired Student t test for comparisonsof two groups and one-way ANOVAwith Tukey post-test for threegroups. P values for the survival curves have been calculated usinga log-rank test. Mann–Whitney U tests were used to assess corre-lation of PD-L1 and T-cell markers in human tumor samples withLKB1 status. Multivariate testing of The Cancer Genome Atlas(TCGA) data with respect to genotype and clinical factors (sex,

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primary tumor stage, nodal stage, metastasis stage, overall stage,age, and smoking) was performed using one-way ANOVA.

ResultsLkb1-deficient tumor cells stimulate neutrophil recruitmentthrough the production of cytokines and chemokines

We previously showed that oncogene activation contributes toescape from immune surveillance by modulating the tumormicroenvironment (10). However, the loss of tumor suppressorshas not been previously investigated in this context. To elucidatehow Lkb1 deficiency impacts the immune microenvironment inlung tumors, we compared the immune cell populations andcytokine profiles ofKras andKras/Lkb1mousemodelswith similardegrees of tumor burden (Supplementary Fig. S1A). We foundthat Lkb1-deficient tumors showed a greater variation in thenumber of total hematopoietic (CD45þ) cells (SupplementaryFig. S1B) and an increase in the total CD11bþ myeloid cellpopulation among three major clusters (CD11c�CD11b�,CD11cþCD11b�, CD11bþ) in the lung (Fig. 1A). Detailed anal-ysis of these myeloid cell populations showed that totalnumbers of TAN (CD11bþLy-6Gþ) are significantly elevatedand tumor-associated alveolar macrophages (TAM: CD11cþ

CD11b�CD103�) are significantly decreased in Kras/Lkb1 tumorscompared withKras tumors (Fig. 1A).Minormyeloid cell popula-tions including eosinophils, Ly-6Chi inflammatory monocytes,and CD103þ dendritic cells did not show significant differences(Supplementary Fig. S1C). Interestingly, the increase of neutro-phils was also observed in the spleen and peripheral blood ofKras/Lkb1 mice (Fig. 1B).

To identify the cytokines and chemokines driving the immunephenotype of Lkb1-deficient tumors, we sorted CD45�EpCAMþ

cells from Kras and Kras/Lkb1 lung tumors using FACS andperformed mRNA sequencing. We discovered higher expressionof a number of chemokines in the Kras/Lkb1 tumor cells: Ppbp[proplatelet basic protein: chemokine (C-X-C motif) ligand 7(Cxcl7)], Cxcl3, and Cxcl5, all of which act through chemokinereceptor CXCR2 on neutrophils (Supplementary Methods), andcytokines: Csf3 [colony stimulating factor 3: granulocyte colonystimulating factor (G-Csf)] and two of the IL1 family of proin-flammatory cytokines, Il33 and Il1a (Fig. 1C).On the contrary, weidentified a decrease in the expression of chemokine (C-C motif)ligand 5 (Ccl5) and Cxcl12 in Kras/Lkb1 tumor cells as comparedwith Kras. Both of these chemokines play an important role inrecruiting lymphocytes and dendritic cells (20, 21) and these celltypes are underrepresented in Kras/Lkb1 tumors (SupplementaryFig. S1C and S1D).

In addition to tumor cells, we sorted TAN from Kras/Lkb1tumors and compared gene expression profiles with TAN fromthe uninduced normal lung from mice with the same geneticbackground. Analysis of mRNA sequencing revealed that TANfrom the Kras/Lkb1 tumors produced elevated T-cell–suppressivefactors (22) including Il10, Lgals9, Arginase 1 (Arg1), and Milk fatglobulin EGF factor 8 protein (Mfge8) and the tumor-promotingcytokine, Il6, as compared with neutrophils from normal lung(Supplementary Fig. 2A).

To confirm these findings at the protein level, we analyzedCXCL7, G-CSF, and IL1a in culture supernatants from cell linesderived frommouse tumors (16). There was a significant increasein CXCL7 and G-CSF in Kras-mutated Tp53-deficient Lkb1-defi-cient cell lines (KPL) compared with Kras-mutated Tp53-deficient

Lkb1-wild type cell lines (KP; Fig. 1D) but IL1a was under thedetection limit in all cell lines (data not shown). In addition to thecytokines identified as differentially expressed inmRNA sequenc-ing, we analyzed IL6 and IL17, two well-characterized cytokinesthat contribute to neutrophil accumulation and production(23, 24), and found that IL6 was significantly increased in KPLcompared with KP (Fig. 1D), but IL17 was not detected (data notshown).Of the cytokines in bronchoalveolar lavagefluids (BALF),CXL7 showed significant difference in Kras/Lkb1 versus controland G-CSF, MFG-E8, and IL10 in Kras/Lkb1 versus control andKras (Fig. 1E and Supplementary Fig. S2B).While Il6 upregulationwas not apparent at themRNA level in EpCAMþKras/Lkb1 tumorscells (data not shown), we detected the cytokine in BALFs fromKras/Lkb1 lungs and in cultured sorted CD45�EpCAMþ cells andTAN from Kras/Lkb1 tumors (Supplementary Fig. S2C). Consid-ering the higher number of neutrophils in Kras/Lkb1 tumors ascomparedwithKras tumors, TANs likely play an important role inaberrant production of this cytokine in addition to tumor cells.Given that IL6 mediates its downstream effects through STAT3(25), wemeasured the levels of phosphorylated STAT3.We foundthat Kras/Lkb1 tumor tissue had higher levels of phospho-STAT3(pSTAT3) than the Kras tumors (Fig. 1F). These findings suggestthat Lkb1 inactivation is associated with neutrophil accumulationto the immunemicroenvironment and overproduction of tumor-promoting cytokines.

In concordance with RNA sequencing data, IL1a showed asignificant increase in BALFs in Kras/Lkb1 versus that from control(Fig. 1G).Mouse BALFs, similar to the supernatants from culturedcells, did not have detectable IL17 (data not shown). To assesswhether IL1a might promote feed forward cytokine signaling inLkb1-deficient tumors, we stimulated a Kras-mutated, p53-loss,Lkb1-lossmouse lung cancer cell line (KPL cell line)with IL1a andanalyzed cytokine secretion. Among the cytokines, we detected anincrease in IL6, CXCL7, and G-CSF production in a dose-depen-dent manner (Fig. 1H and Supplementary Fig. S3A). These resultsare consistent with Lkb1-loss increasing IL1a production, whichthen promotes the activation of IL6–STAT3 signaling.

Lkb1 loss negatively impacts the number and function of tumorinfiltrating T cells and PD-L1 expression on tumor cells

Clinical studies have demonstrated that the density of tumor-infiltrating lymphocytes is associated with a favorable prognosisand response of immunotherapy in cancer (7–9). We found thattotal counts of both CD4 and CD8 T cells were significantlydecreased in Kras/Lkb1mouse tumors (Fig. 2A) as compared withKras tumors. Infiltrating T cells showed a significantly higherexpression of T-cell–inhibitory markers: Programmed cell deathprotein 1 (PD-1), T-cell immunoglobulin mucin-3 (TIM-3), Lym-phocyte-activation gene 3 (LAG-3), and CTL-associated protein 4(CTLA-4; Fig. 2B). We also confirmed expression of the ligand forTIM-3, LGALS9, in both tumor cells and TAN from Kras/Lkb1tumors by flow cytometry (Supplementary Fig. S2D). The ratio ofregulatory T cells (FOXP3þ) to total CD4 T cells was also signif-icantly increased in Kras/Lkb1 tumor as compared with Krastumors (Fig. 2B). We evaluated T-cell function in Kras/Lkb1 andKras tumors with similar levels of disease (Supplementary Fig.S3B) and found significantly less IFNg and Ki-67 expression intotal CD4 and CD8 T cells from the Kras/Lkb1 tumors than thosefrom Kras tumors (Fig. 2C). Thus, Lkb1 inactivation is associatedwith reduced T-cell number and increased markers of T-cellexhaustion.

Immunosuppressive Effects of STK11/LKB1 Loss in Lung Cancer

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To understand the role of neutrophils in this model, we used aneutrophil-depleting (anti-Ly-6G/Gr-1:RB6-8C5) antibody inmice with established tumors (Supplementary Fig. S4A andS4B). Kras/Lkb1 mice treated with anti Ly-6G/Gr-1 antibody for1 or 2 weeks showed a significant reduction of TAN and of IL6andG-CSF in BALFs (Supplementary Fig. S4C and S4D), resulting

in a significant increase in total CD8 T-cell numbers, proliferation(Ki-67þ), and T-cell function represented by IFNg production(Supplementary Fig. S4E).

PD-L1 expression on tumor cells is a biomarker associatedwith a response to PD-1 blockade treatment (7, 10). Lkb1-defi-cient tumor cells expressed significantly lower levels of PD-L1 in

Figure 1.Tumor-suppressor Lkb1 inactivation promotes neutrophil accumulation via proinflammatory cytokines and chemokines. A, immune cell populations in thelung tumors from Kras (K) and Kras/Lkb1 (KL) mouse models. Representative flow cytometry data (live/single/total CD45þ cells) from each mousemodel (left). Total counts of TAN: CD11bþLy-6Gþ cells and TAMs: CD11cþCD11b�CD103� fromKras (K; n¼ 8) andKras/Lkb1 (KL; n¼ 8)mice. �� ,P <0.01; �� ,P <0.001.B, neutrophil counts in the spleen and peripheral blood from Kras (n ¼ 8) or Kras/Lkb1 (n ¼ 8) mice (right). �� , P < 0.01. C, expression of immune-modulatingfactors from RNA sequencing of the sorted tumor cells (CD45�EpCAMþ) in Kras (K Ep) or Kras/Lkb1 (KL Ep) mice and uninduced normal lung CD45�EpCAMþ

cells (control). Each column consists of a combination of samples derived from 3 to 4 mice. Log-transformed FPKM values are shown, colored blue/red forlow/high expression, respectively. Epcam and Cd45 expression are shown as positive and negative controls. Differential expression is shown as fold-change values,colored blue/red for under/overexpression compared with controls. D and E, chemokine and cytokine levels in the culture supernatants after 48-hour incubationfrom Kras/p53 (KP; n ¼ 3) versus Kras/p53/Lkb1 (KPL; n ¼ 3) cell lines generated from mouse lung tumors; �� , P < 0.001. Data indicate three replicate wellsand are representative of three independent experiments (D) andBALF from littermate controls (n¼ 5),Krasmice (n¼ 8) orKras/Lkb1mice (n¼ 8; E). � , P <0.05; �� ,P < 0.01 . F, Western blot analysis for pSTAT3, STAT3 levels in Kras versus Kras/Lkb1 tumors. Each column represents tumor from a different mouse and actinrepresents loading control. G, IL1a level in the BALF from C (n¼ 5), Kras (n¼ 8), or Kras/Lkb1 (n¼ 8)mice. � , P < 0.05. H, IL6 levels in culture supernatants measured24 hours after IL1a stimulation (0, 5, and 20 ng/mL) of KP (n ¼ 3) versus KPL (n ¼ 3) cell lines. Data indicate three replicate wells and are representative ofthree independent experiments.

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CD45�EpCAMþ cells as comparedwithKras tumor cells (Fig. 2D).PD-L1 expression is influenced by a variety of factors that includenon–cell-autonomous factors such as release of IFNg from T cells(26) in the tumor microenvironment in vivo. To dissect theintrinsic role of Lkb1 inactivation on PD-L1 expression specificallyin tumor cells, we analyzed PD-L1 expression in cultured cell linesderived frommouse tumors of the KP and KPL genotypes. PD-L1levels were significantly lower in KPL as compared with KP (Fig.2E). To confirm that our findings in mouse models and cell lineswere applicable to humans, we studied human lung cancer celllines with endogenous KRAS mutation and wild-type or inacti-vated LKB1. We either performed knockdown of LKB1 usingshRNA or reconstituted LKB1 with wild-type (WT) or kinase dead(KD) LKB1 to develop isogenic cell lines (wild -type cells: H441,H1792, and LKB1-mutant cell: A549; Supplementary Fig. S5A).PD-L1 expression was lower in both of the LKB1 WT lines whenexpressing sh-LKB1 (Fig. 2F). Expression of LKB1 (WT and KD) inthe LKB1-deficient A549 cell line resulted in a modest increase inPD-L1 levels (Supplementary Fig. S5B). These data suggest LKB1inactivation decreased PD-L1 levels independent of IFNg .

Functional loss of LKB1 in human cell lines is phenotypicallysimilar to mouse Kras/Lkb1 tumors

We next assessed the cytokine and chemokine profiles inhuman isogenic cell lines to determine whether similar patternswould be observed. We analyzed culture supernatants from thesecell lines and found that IL6 and G-CSF were significantlyincreased in LKB1-inactivated cells as compared with LKB1-intactcells (Fig. 3A and Supplementary Fig. S5C). There was also asignificant increase of CXCL7 that was only detected in A549 cells(Supplementary Fig. S5C). IL1a stimulation of these cell lines ledto an increase in IL6, G-CSF, and CXCL7 in a dose-dependentmanner (Fig. 3B and Supplementary Fig. S5D), which was con-sistent with the Lkb1-deficient mouse cell line data (Fig. 1D). Wealso found that IL6 induction by IL1a stimulation was morepronounced in LKB1-deficient cell lines as compared with LKB1-intact cell lines (Supplementary Fig. S5E) except forH1792,whichshowed modest stimulation. This line has high baseline IL1aproduction (Fig. 3A) and the shRNA only partially knocked downLKB1 (Supplementary Fig. S5A), which makes this result difficultto interpret.

Figure 2.Lkb1 inactivation leads to a T-cell–suppressive tumor microenvironment with low PD-L1 expression in tumor cells. A, total counts of CD4 T cells (top) and CD8 T cells(bottom). ��, P < 0.01. B, expression of checkpoint receptors in CD4 T cells (top) and CD8 T cells (bottom) in Kras (K) or Kras/Lkb1 (KL) tumors. � , P < 0.05;�� , P < 0.01; �� , P < 0.001. C, IFNg expression and proliferation marker (Ki-67) positivity for CD4 or CD8 T cells in Kras or Kras/Lkb1 tumors. Representative flowcytometry data (total CD3þ T cells) from each mouse model (left). Percentage of Ki-67þ and IFNgþ in CD4 or CD8 T cells from Kras (n ¼ 6) or Kras/Lkb1(n ¼ 6) mice. �, P < 0.05; �� , P < 0.01. D, PD-L1 expression in gated CD45�EpCAMþ cells in Kras (n ¼ 5) or Kras/Lkb1 (n ¼ 5) tumors evaluated by flowcytometry. � , P¼ 0.0384. E, PD-L1 expression in KP (n¼ 3) versus KPL (n¼ 3) cell lines evaluated by flow cytometry. � , P¼ 0.0495. Data are representative of threeindependent experiments. F, PD-L1 expression in H441 or H1792 cells stably transfected with sh-nontarget (NT) or sh-LKB1. Data are representative of threeindependent experiments.



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Analysis of lung cancer cell lines from The Cancer Cell LineEncyclopedia (CCLE; ref. 27) confirmed that PD-L1 expressionis significantly lower in cell lines with LKB1 mutation (32 LKB1WT and 4 LKB1-mutant cell lines; Fig. 3C), although the smallnumber of LKB1-mutant lines precluded any significant associa-tions among the immune-related genes displayed in Fig. 1C andSupplementary Fig. S2A and LKB1 status when corrected formultiple hypotheses. In addition, analysis of KRAS-mutated lungadenocarcinomas from The Cancer Genome Atlas (TCGA-52LKB1WT, 15 LKB1mutant; ref. 2) showed that PD-L1 expressionwas significantly reduced in LKB1-mutatedNSCLCs (Fig. 3C). In a

multivariate analysis of the TCGA dataset with respect to LKB1status and clinical factors, PD-L1 and LKB1 status were alsosignificantly associated (P ¼ 0.005). To validate these findingsin an independent dataset, PD-L1 mRNA was assessed in the MDAnderson PROSPECT (Profiling of Resistance Patterns and Onco-genic Signaling Pathways in Evaluation of Cancers of the Thoraxand Therapeutic Target Identification) cohort (MDACC-108 LKB1WT, 44 LKB1-MUTANT cases). We again observed an associationamong PD-L1 expression and LKB1 status. We also validated andquantitated the expressionof PD-L1 at the protein level by reverse-phase protein arrays (RPPA) in 106 cases from theMDACCcohort

Figure 3.LKB1 inactivation in human KRAS-mutated cell lines showed similar phenotype with mouse Kras/Lkb1 tumor. A, analysis of IL6 in the culture supernatantsafter 48-hour incubation of KRAS-mutated LKB1 wild-type H441 or H1792 cells stably transfected with sh-NT or sh-LKB1 and KRAS, LKB1-mutant A549 cellsreconstituted with empty vector (Vector), wild-type LKB1, or kinase dead LKB1 (LKB1KD). �, P < 0.05; ��, P < 0.001. Data indicate three replicate wells and arerepresentative of three independent experiments. B, IL6 levels in culture supernatants measured 24 hours after IL1a stimulation (0, 5, and 20 ng/mL) ofthree LKB1-deficient cell lines. Data indicate three replicate wells and are representative of three independent experiments. C, PDL1 expression in KRAS (K) or KRASand LKB1 mutated (KL) cell lines from CCLE database (�, P ¼ 0.04) and PDL1 expression in Kras (K) or Kras/Lkb1 (KL) lung adenocarcinoma samples fromTCGA database (�� , P ¼ 0.00004). D, PD-L1 mRNA levels determined by microarray (P ¼ 0.1) and protein levels (�� , P ¼ 0.009) determined by RPPA fromthe MDACC dataset. E, representative IHC for PD-L1 on the KRAS-mutated LKB1 wt or mutant patient tumors from the MDACC cohort. F, CD3 (�� , P ¼ 0.002)-and CD8 (�� , P ¼ 0.0003)-positive cell densities by IHC on the MDACC patient cohorts.

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and detected significantly lower levels of PD-L1 in LKB1-mutanttumors (Fig. 3D). As the current clinical standard detectionmethod for the PD-L1 expression is IHC, we confirmed thedifference in PD-L1 expression by IHC (Fig. 3E).

Next, to evaluate the functional effect of LKB1-loss in the tumormicroenvironment in patient tumors, we analyzed the T-cellinfiltrate in the tumors by IHC. In 19 LKB1 WT and 11 LKB1-mutant tumors, total T-cell (CD3þ) and CD8 T-cell counts anddensities were significantly lower in LKB1-inactivated tumors ascompared with LKB1-intact tumors (Fig. 3F), In sum, observa-tions in patient cell lines and tumor samples are consistent withour findings in Kras/Lkb1 mice, suggesting that LKB1 mutationnegatively regulates PD-L1 expression and reduces CD8 T-cellinfiltration.

Neutralizing IL6 leads to a therapeutic benefit inKras/Lkb1miceSupporting the notion that PD-L1 expression in tumor cells is

critical in the response to PD-1 blockade, treatment of the Kras/

Lkb1mousemodel with a PD-1 blocking antibody did not show asignificant treatment response (Fig. 4A). Given our observation ofelevated IL1a and IL6 in BALFs (Fig. 1E and G) and aberrantactivation of pSTAT3 in tumor nodules (Fig. 1F) from Kras/Lkb1mice as compared with Krasmice, we hypothesized that targetingaberrant cytokine production could be a rational therapeuticstrategy in Kras/Lkb1–mutant tumors. To evaluate the in vivoefficacy of IL6 blockade, we treated Kras/Lkb1 mice with a neu-tralizing IL6 antibody (MP5-20F3). The therapeutic anti-IL6 anti-body significantly inhibited tumor progression as compared withanti PD-1 antibody (Fig. 4A). In addition, IL6 antibody–treatedmice showed significantly improved survival as compared withcontrol mice (Fig. 4B). However, treatment of Kras/Lkb1 tumorswith other checkpoint-blocking antibodies against CTLA-4 or acombination of PD-1 and TIM-3 also did not demonstrate anyefficacy (data not shown). Taken together, these findings suggestthat Lkb1-loss results in a T-cell–suppressed environment as aconsequence of autocrine and neutrophil-induced cytokine

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Figure 4.IL6-neutralizing treatment showed clinical efficacy in Kras/Lkb1 mouse model. A, representative images of MRI and quantification of MRI from KL micetreated with PD-1 or IL6-blocking antibodies or controls. B, survival of untreated mice versus mice treated with IL6-blocking antibody (�� , P¼ 0.0002, n¼ 6 vs. 12,respectively). C and D, IL6 and G-CSF levels in BALFs (C) and TAN counts (D) for untreated KL mice (n ¼ 7) or KL mice treated with IL6-neutralizingantibody (n¼ 8), with comparable tumor burden. � , P <0.05; �� , P <0.01. E, Ki-67 and IFNg-positive CD8 T-cell counts in untreated KLmice (n¼ 7) or KLmice treatedwith IL6-neutralizing antibody (n ¼ 8), with comparable tumor burden. F, representative Ki-67 and TUNEL IHC and quantification per the microscopic fieldon the KL mice untreated or treated with IL6-neutralizing antibody. Each data point represents a different microscopic field. For Ki-67, n ¼ 9 and 5 and for TUNEL,n ¼ 8 and 5 for untreated and IL6 antibody (ab)-treated mice, respectively. �� , P ¼ 0.0049 for Ki-67; �� , P ¼ 0.0024 for TUNEL.



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production and not engagement of the PD-L1:PD-1 immunecheckpoint.

To further investigate the effect of IL6–neutralizing antibody onthe immune profile of Kras/Lkb1 tumors, we treated themice for 2weeks and then performed immune and histologic analyses(Supplementary Fig. S6A). There was a significant reduction indetectable IL6 as expected as well as G-CSF in BALFs from thetreated mice (Fig. 4C). In keeping with the neutrophil-attractingcytokines and chemokines observed in BALFs, there was a signif-icant reduction in the counts of total TAN with IL6 neutralization(Fig. 4D), resulting in functional recovery of T cells (Fig. 4E).Treated tumors also exhibited elevation of CD4 T cells, CD8 Tcells, and TAM to levels that are comparable with the Lkb1 wild-type tumors (Supplementary Fig. S6B). In addition to theimmune-related effects, IL6 antibody–treated tumor cells exhib-ited significantly less proliferation and increased apoptosis (Fig.4F). Although therapeutic IL6 blockade improved T-cell function,concurrent therapy combining anti-IL6 and PD-1 treatment didnot demonstrate additional benefit as compared with IL6 block-ade alone in terms of survival (Supplementary Fig. S6C). Thissuggests a need to further define contexts in which cytokinesuppression and immune checkpoint blockade might be utilizedtogether to enhance therapeutic benefit as compared with eithertreatment alone.

DiscussionOncogenes and tumor suppressors promote self-sufficient sig-

naling for autonomous proliferation of tumor cells. Recent workhas shown that oncogenic mutations alter the tumor microenvi-ronment, cause immune suppression, and can impact theresponse to immune-modulating treatment strategies (10).Somatic mutations also produce neoantigens, which are recog-nized by the immune system and mediate sensitivity of thetumors to immunotherapies (28–30). Here, we have shown thatinactivation of the tumor suppressor gene STK11/LKB1 causesdramatic changes in the tumor microenvironment in addition tothe previously reported effects on cell cycle, metabolism, differ-entiation, polarity, and other cellular pathways (16, 31).

We have shown that LKB1 inactivation promoted the produc-tion of proinflammatory cytokines CXCL7, G-CSF, and IL6 inboth mouse tumors and cell lines, which contributes to neutro-phil accumulation. Elevation of the proinflammatory cytokineIL1a was confirmed in vivo but not robustly in cell culture super-natants from Lkb1-deficient cells. Previous studies have shownthat IL1a is released only under specific conditions includingnecrotic cell death and inflammasome activation (32, 33), sug-gesting that the release of IL1a could be caused by necrotic celldeath in Kras/Lkb1 tumor microenvironment and that this facil-itates the activation of IL6–STAT3 signaling pathway in Kras/Lkb1tumors together with IL6, producing neutrophil accumulation.

The tumor microenvironment in Kras/Lkb1 tumors displayedcharacteristics of T-cell suppression with fewer lymphocytes,higher levels of checkpoint receptor expression in those, and anincrease in TANs with suppressive properties compared with Krastumors. TANs expressing high levels of Il10, Arginase1, andMfge8that have been implicated in T-cell suppression and Treg induc-tion (15, 22, 34). Depleting TAN using an anti Ly-6G/Gr-1antibody improved T-cell function. Although the role of TAN insuppressing T-cell function is controversial (35), TAN appear toact in an immunosuppressive fashion in this context, suggesting

that therapies suppressing TAN should be further explored asimmunomodulatory therapies.

Moreover, we found that Lkb1 inactivation caused a decrease inPD-L1 levels on tumor cells from Kras/Lkb1 tumors and incultured cells frommice andpatients. Conforming to the previousobservations proposing an association of tumor cell PD-L1expression, the magnitude of T-cell accumulation in the tumorsand response to PD-1 blockade (7, 8, 36), treatment with a PD-1–blocking antibody did not show efficacy in the treatment of theKras/Lkb1 mouse model. A recent study (30) as well as our owninstitutional experience suggest thatwhileKRAS-mutated patientsrespond favorably to PD-1 blockade (PFS of 15 months onpembrolizumab) this is not seen in patients with LKB1mutationsalthough large cohorts will be needed to define genotype–response associations in detail. In addition, contributing to lowPD-1:PD-L1 levels inKras/Lkb1 tumors is the greater proportion ofTAN that express low levels of PD-L1 as compared with TAM. Thecoordinated role of PD-L1 expression in the tumor cells and thecells constituting the immune microenvironment in this modelrequires further study to define improved immunotherapeuticstrategies.

Treatment of Kras/Lkb1 tumors with an IL6–blocking anti-body decreased tumor cell proliferation and increased T-cellfunction, resulting in a therapeutic effect in Kras/Lkb1 mousemodel while immune checkpoint blockade was not efficacious.These data suggest that mouse models can be used to model thetumor microenvironment and predict response to novelimmune-modulating treatment strategies based on rationalpredictions from studies of the tumor microenvironment.Future studies will examine whether cytokines other than IL6also contribute to STAT3 activation (37), and whether com-bined cytokine blockade (e.g., with IL1 neutralization) will leadto more durable therapeutic effects. Although we found thatneutralizing IL6 antibodies improved T-cell number and func-tion in Lkb1-deficient tumors, the combination of anti PD-1plus anti IL6 antibodies did not improve outcome when givenconcurrently. There may be technical limitations to this con-sidering both of the antibodies are Rat IgG isotype and com-bination treatment can lead to antibody neutralization. Fur-thermore, dosing schedules of cytokine suppression and com-binations with other therapeutic agents such as immune check-point blockade will need to be studied.

In summary, we have presented a novel set of findings thatsuggest that not only oncogene driver mutations but also tumorsuppressor gene mutations can modify the immune microenvi-ronment in lung cancer. In this example, focusing on Lkb1 loss, weobserved a marked increase in inflammatory cytokines thatrecruited neutrophils and inhibited the function of T cells. Wealso showed that PD-1 checkpoint blockade was ineffective inLkb1-mutant cancers, whereas targeting IL6 displayed a significantalbeit short-lived treatment response in the Kras/Lkb1 model.These findings suggest that IL6-dependent signaling activationcan be a therapeutic target in Lkb1-deficient Kras-driven lungtumors and potentially other tumors with high levels of IL6, andalso suggest targeting aberrant inflammation by inhibiting cyto-kine signaling may represent a promising immunotherapeuticstrategy in selected patients.

Disclosure of Potential Conflicts of InterestM. Hellmann reports receiving a commercial research grant from Bristol-

Myers Squibb and is a consultant/advisory board member for Bristol-Myers

Koyama et al.


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Squibb, Genentech, Third Rock Ventures, and Inovio Pharmaceuticals. J.V.Heymach reports receiving other commercial research support from Astra-Zeneca, GlaxoSmithKline, Bayer and is a consultant/advisory board memberfor Genentech, AstraZeneca, Novartis, GlaxoSmithKline, Lilly, BoerhingerIngelheim, Synta, and Exelixis. F.S. Hodi is a consultant in Merck and reportsreceiving a commercial research grant from Bristol Myers Squibb and Merck(to institution). G.J. Freeman has ownership interest (including patents) inMerck, Bristol Myers Squibb, Roche (immediate family member), EMDSerono, Amplimmune, Boehringer Ingelheim, Novartis (immediate familymember) and is a consultant/advisory board member for Novartis (imme-diate family member), Roche, Bristol Myers Squibb, Eli Lilly, and SurfaceOncology (immediate family member). D.A. Barbie is a consultant/advisoryboard member for N-of-One. G. Dranoff is a global head, exploratoryimmune-oncology in Novartis. No potential conflicts of interest were dis-closed by the other authors.

Authors' ContributionsConception and design: S. Koyama, E.A. Akbay, G.S. Herter-Sprie, P.E. Fecci,J.V. Heymach, G. Dranoff, P.S. Hammerman, K.-K. WongDevelopment of methodology: S. Koyama, E.A. Akbay, A.R. Aref, F. Skoulidis,G.J. FreemanAcquisition of data (provided animals, acquired and managed patients, pro-vided facilities, etc.): S. Koyama, E.A. Akbay, F. Skoulidis, G.S. Herter-Sprie,K.A. Buczkowski, Y. Liu, M.M. Awad, E.R. Parra Cuentas, I.I. Wistuba, M. Sou-cheray, H. Asahina, S. Kitajima, A. Altabef, J.D. Cavanaugh, K. Rhee, P.E. Fecci,T. Shimamura, M. Hellmann, J.V. Heymach, F.S. Hodi, D.A. Barbie, K.-K. WongAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): S. Koyama, E.A. Akbay, Y.Y. Li, F. Skoulidis,G.S. Herter-Sprie, K.A. Buczkowski, Y. Liu, M.M. Awad, W.L. Denning, L. Diao,J. Wang, E.R. Parra Cuentas, I.I. Wistuba, S. Kitajima, F.S. Hodi, D.A. Barbie,G. Dranoff, P.S. HammermanWriting, review, and/or revision of the manuscript: S. Koyama, E.A. Akbay,Y.Y. Li, F. Skoulidis, G.S. Herter-Sprie, K.A. Buczkowski, H. Zhang, M. Hellmann,J.V. Heymach, F.S. Hodi, G.J. Freeman, G.Dranoff, P.S. Hammerman, K.-K.WongAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.S. Herter-Sprie, Y. Liu, K. Rhee, P. Gao,H. Zhang, J.V. Heymach, G.J. Freeman, P.S. Hammerman

Study supervision: D.A. Barbie, G. Dranoff, P.S. Hammerman, K.-K. WongOther (pathology involving in the data analysis): E.R. Parra CuentasOther (Western blot analysis): T. ThaiOther (construction of cell line models): T. Shimamura

AcknowledgmentsThe authors thank Suzan Lazo-Kallanian, John Daley, Kristen Cowens,

and Steven Paul for help with flow cytometry anlaysis, Christine Lam fortissue processing, Mei Zhang for IHC, Xiaoen Wang for helping with mousestudies, and the Dana Farber Center for Cancer Genome Discovery for RNAsequencing.

Grant SupportP.S. Hammerman is supported by a Clinical Investigator Award

from the Damon Runyon Cancer Research Foundation, a DevelopmentalProject Grant from NCI P50 CA090578 and the Starr Consortium forCancer Research. K.-K. Wong is supported by NIH/NCI P01 CA120964,5R01CA163896-04, 1R01CA195740-01, 5R01CA140594-07, 5R01CA122794-10, and 5R01CA166480-04 grants and support from Gross-Loh Family Fund forLung Cancer Research and Susan Spooner Family Lung Cancer Research Fund atDana-Farber Cancer Institute. P.S. Hammerman, K.K. Wong, J.V. Heymach, andM.D. Hellman are supported by a Stand Up To Cancer - American Cancer SocietyLung Cancer Dream Team Translational Research Grant (Grant Number: SU2C-AACR-DT17-15). StandUp ToCancer is a program of the Entertainment IndustryFoundation. Research grants are administered by the American Association forCancer Research, the scientific partner of SU2C. S. Koyama is supported byMargaret A. Cunningham Immune Mechanisms in Cancer Research FellowshipAward and The Kanae Foundation for the Promotion of Medical Science Fellow-ship Award. G.S. Herter-Sprie was supported by the Deutsche Forschungsge-meinschaft (HE 6897/1-1) and the Claudia Adams Barr Program for InnovativeCancer Research. T. Shimamura is supported by Uniting Against Lung CancerLegacy Program and American Cancer Society Research Scholar Award. G.J.Freeman is supported by NIH R01AI08995.

Received May 29, 2015; revised November 24, 2015; accepted December 6,2015; published OnlineFirst February 1, 2016.

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